Purification and characterization of a thermostable intra-cellular β-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus

An intra-cellular β-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatography and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited α-helical conformation. MALDI-TOF identified the enzyme’s molecular weight as 6688 Daltons, but SDS–PAGE and immunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique properties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 °C and residual activity was 80–100% between pH ranges 5–8. The enzyme had higher specific activity against p-nitrophenyl-d-glucopyranoside than cellobiose and HPLC showed that the enzyme possesses transglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzyme will be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical hazards. This is the first report of a β-glucosidase enzyme with such a low monomeric unit size.